Clearcoat containing thermoase c160 for easy-cleaning of insect body stains

ABSTRACT

A substrate or coating is provided that includes a protease with enzymatic activity toward a component of a biological stain. Also provided is a process for facilitating the removal of a biological stain is provided wherein an inventive substrate or coating including a protease is capable of enzymatically degrading of one or more components of the biological stain to facilitate biological stain removal from the substrate or said coating.

FIELD OF THE INVENTION

The present invention relates generally to coating compositionsincluding bioactive substances and methods of their use to facilitateremoval of insect stains. In specific embodiments, the invention relatesto compositions and methods for prevention of insect stain adherence toa surface as well as insect stain removal by incorporating a proteaseinto or on polymer composite materials to degrade insect bodycomponents.

BACKGROUND OF THE INVENTION

Many outdoor surfaces are subject to stain or insult from naturalsources such as bird droppings, resins, and insect bodies. As a result,the resulting stain often leaves unpleasant marks on the surfacedeteriorating the appearance of the products.

Traditional self-cleaning coatings and surface are typically based onwater rolling or sheeting to carry away inorganic materials. These showsome level of effectiveness for removal of inorganic dirt, but are lesseffective for cleaning stains from biological sources, which consist ofvarious types of organic polymers, fats, oils, and proteins each ofwhich can deeply diffuse into the subsurface of coatings. Prior artapproaches aim to reduce the deposition of stains on a surface andfacilitate its removal capitalize on the “lotus-effect” wherehydrophobic, oleophobic and super-amphiphobic properties are conferredto the surface by polymeric coatings containing appropriatenanocomposites. An exemplary coating contains fluorine and siliconnanocomposites with good roll off properties and very high water and oilcontact angles. When used on rough surfaces like sandblasted glass,nanocoatings may act as a filler to provide stain resistance. A drawbackof these “passive” technologies is that they are not optimal for use inhigh gloss surfaces because the lotus-effect is based on surfaceroughness.

Photocatalytic coatings are promising for promoting self-cleaning oforganic stains. Upon the irradiation of sun light, a photocatalyst suchas TiO₂ chemically breaks down organic dirt that is then washed away bythe water sheet formed on the super hydrophilic surface. As an example,the photocatalyst TiO₂ was used to promote active fingerprintdecomposition of fingerprint stains in U.S. Pat. Appl. Publ.2009/104086. A major drawback to this technology is its limitation touse on inorganic surfaces due to the oxidative impairment of the polymercoating by TiO₂. Also, this technology is less than optimal forautomotive coatings due to a compatibility issue: TiO₂ not onlydecomposes dirt, but also oxidizes polymer resins in the paint.

Therefore, there is a need for new materials or coatings that canactively promote the removal of biological stains on surfaces or incoatings and minimize the requirement for maintenance cleaning.

SUMMARY OF THE INVENTION

A process of facilitating the removal of biological stains is providedincluding providing a substrate or a coating with an associated proteaseor analogue thereof such that said protease or analogue thereof iscapable of enzymatically degrading a component of a biological stain. Aprotease is optionally a member of the M4 thermolysin-like proteaseswhich include thermolysin or analogues thereof. In some embodiments aprotease is a bacterial neutral thermolysin-like-protease from Bacillusstearothermophilus or an analogue thereof.

The surface activity is related to the ability of the substrate orcoating to facilitate biological stain removal. The substrate or coatingoptionally has a surface activity that is 0.0075 units/cm² or greater.

A protease or analogue thereof is optionally covalently attached,non-covalently adhered to or admixed into to the substrate or to thecoating or combinations thereof.

A substrate or coating optionally includes an organic crosslinkablepolymer resin. An organic crosslinkable polymer resin optionallyincludes a functional group of acetoacetate, acid, amine, carboxyl,epoxy, hydroxyl, isocyanate, silane, vinyl, or combinations thereof. Insome embodiments the organic crosslinkable polymer resin includesaminoplasts, melamine formaldehydes, carbamates, polyurethanes,polyacrylates, epoxies, polycarbonates, alkyds, vinyls, polyamides,polyolefins, phenolic resins, polyesters, polysiloxanes, or combinationsthereof. Optionally, an organic crosslinkable polymer is ahydroxyl-functionalized acrylate resin.

Also provided is a composition for facilitating biological stain removalincluding a substrate or a coating and a thermolysin, thermolysin-likeprotease, or analogues thereof capable of degrading a biological staincomponent, wherein the thermolysin, thermolysin-like protease, oranalogue thereof is associated with the substrate or coating. Thethermolysin-like protease is optionally a bacterial neutralthermolysin-like-protease from Bacillus stearothermophilus or analoguethereof. In some embodiments the substrate or said coating has a surfaceactivity of 0.0075 units/cm² or greater.

A composition optionally includes an organic crosslinkable polymerresin. An organic crosslinkable polymer resin optionally includes afunctional group of acetoacetate, acid, amine, carboxyl, epoxy,hydroxyl, isocyanate, silane, vinyl, or combinations thereof. In someembodiments the organic crosslinkable polymer resin includesaminoplasts, melamine formaldehydes, carbamates, polyurethanes,polyacrylates, epoxies, polycarbonates, alkyds, vinyls, polyamides,polyolefins, phenolic resins, polyesters, polysiloxanes, or combinationsthereof. Optionally, an organic crosslinkable polymer is ahydroxyl-functionalized acrylate resin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents a schematic of a spray-down application process of oneembodiment of a coating;

FIG. 2 represents a schematic of stain application and protease mediatedstain removal on a coated substrate;

FIG. 3 illustrates improved rinsing of insect stains using a B.stearothermophilus TLP (Thermoase C160) based coating relative to anenzyme free control;

FIG. 4 illustrates improved rinsing of insect stains using a B.stearothermophilus TLP (Thermoase C160) based coating relative to aLipase PS based coating;

FIG. 5 illustrates improved rinsing of insect stains using a B.stearothermophilus TLP (Thermoase C160) based coating relative to anα-Amylase based coating;

FIG. 6 illustrates affects on 100° C. baking for 10 days on surfaceenzyme activity (A) and stain cleaning time (B);

FIG. 7 illustrates increased loading of protease increases self-cleaningperformance with relative enzyme loading concentrations of 0.2% (A),2.0% (B), 4.0% (C), 6.0% (D), and 8.0% (E);

FIG. 8 illustrates rinsing of insect stains by a (A) Protease N based SBcoating, (B) Protin SD AY-10 based SB coating, (C) Protease A based SBcoating, (D) Thermoase GL30 based SB coating (<0.0075 units/cm² surfaceactivity B. stearothermophilus TLP), or (E) Thermoase C160 based SBcoating;

FIG. 9 illustrates a schematic of a road test protocol for active stainremoval by an embodiment of a coating;

FIG. 10 illustrates rain or water bath rinsing of enzyme containing orcontrol coatings after depositing insect bodies during road driving;

FIG. 11 illustrates rain or water bath rinsing of enzyme containing orcontrol coatings after depositing insect bodies during road driving, WBSrepresents enzyme coatings; WBB represents control coatings; (A) leftpanes are before rinsing, (B) middle panel after 5 min of rinsing, (b) 2hours of rinsing; blue circles are counted stains; red lines are theboundaries of the counting area;

FIG. 12 illustrates average road obtained insect stain removal frompanels coated with an enzyme containing coating or a control coating;

FIG. 13 illustrates average road obtained insect stain removal frompanels coated with an enzyme containing coating or a control coatingwhereby the enzyme containing coatings are prepared at different bufferpH levels.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of embodiment(s) of the invention is merelyexemplary in nature and is in no way intended to limit the scope of theinvention, its application, or uses, which may, of course, vary. Theinvention is described with relation to the non-limiting definitions andterminology included herein. These definitions and terminology are notdesigned to function as a limitation on the scope or practice of theinvention but are presented for illustrative and descriptive purposesonly.

The present invention is based on the catalytic activity of a proteaseenzyme to selectively degrade components of organic stains thus,promoting active stain removal. Organic stains typically include organicpolymers, fats, oils, and proteins. It was traditionally difficult toidentify a protease that was simultaneously incorporatable into or on acoating or substrate with remaining activity and successfully promoteactive breakdown and subsequent removal of biological stains,particularly stains from insect sources. The inventors unexpectedlydiscovered that a particular family of hydrolases, the bacterialthermolysins (EC 3.4.24.27) successfully promoted biological stainremoval whereas similar proteases, even other closely relatedmetalloproteases, were unsuccessful.

The protease is either immobilized into or on coatings or substrates andcatalyzes the degradation of biological stain components into smallermolecules. The small product molecules are less strongly adherent to asurface or coating such that gentle rinsing such as with water, air, orother fluid promotes removal of the biological material from the surfaceor coating. Thus, the invention has utility as a composition and methodfor the active removal of biological stains from surfaces.

It is appreciated that the while the description herein is directed tocoatings, the materials described herein may also be substrates orarticles that do not require a coating thereon for promotion offunctional biological stain removal. As such, the word “coating” as usedherein means a material that is operable for layering on a surface ofone or more substrates, or may comprise the substrate material itself.As such, the methods and compositions disclosed herein are generallyreferred to as a protease associated with a coating for exemplarypurposes only. One of ordinary skill in the art appreciates that thedescription is equally applicable to substrates themselves.

An inventive method includes providing a coating with a protease suchthat the protease is enzymatically active and capable for degrading oneor more components of a biological stain. In particular embodiments abiological stain is based on bioorganic matter such as that derived froman insect, optionally an insect body.

A biological stain as defined herein is a bioorganic stain, mark, orresidue left behind after an organism contacts a substrate or coating. Abiological stain is not limited to marks or residue left behind after acoating is contacted by an insect body. Other sources of bioorganicstains are illustratively: insect wings, legs, or other appendages; birddroppings; fingerprints or residue left behind after a coating iscontacted by an organism; or other sources of biological stains.

A protease is optionally a bacterial metalloprotease such as a member ofthe M4 family of bacterial thermolysin-like proteases of whichthermolysin is the prototype protease (EC 3.4.24.27) or analoguesthereof. A protease is optionally the bacterial neutralthermolysin-like-protease (TLP) derived from Bacillus stearothermophilus(Bacillus thermoproteolyticus Var. Rokko) (illustratively sold under thetrade name “Thermoase C160” available from Amano Enzyme U.S.A., Co.(Elgin, Ill.)) or analogues thereof. A protease is optionally anyprotease presented in de Kreig, et al., J Biol Chem, 2000;275(40):31115-20, the contents of which are incorporated herein byreference. Illustrative examples of a protease include thethermolysin-like-proteases from Bacillis cereus (Accession No. P05806),Lactobacillis sp. (Accession No. Q48857), Bacillis megaterium (AccessionNo. Q00891), Bacillis sp. (Accession No. Q59223), Alicyclobacillisacidocaldarious (Accession No. Q43880), Bacillis caldolyticus (AccessionNO. P23384), Bacillis thermoproteolyticus (Accession No. P00800),Bacillus stearothermophilus (Accession No. P43133), Bacillus subtilis(Accession No. P06142), Bacillus amyloliquefaciens (Accession No.P06832), Lysteria monocytogenes (Accession No: P34025; P23224), amongothers known in the art. The sequences at each accession number listedherein are incorporated herein by reference. Methods of cloning,expressing, and purifying any protease operable herein is achievable bymethods ordinarily practiced in the art illustratively by methodsdisclosed in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989; Current Protocols in Molecular Biology, ed. Ausubelet al., Greene Publishing and Wiley-Interscience, New York, 1992 (withperiodic updates); and Short Protocols in Molecular Biology, ed. Ausubelet al., 52 ed., Wiley-Interscience, New York, 2002, the contents of eachof which are incorporated herein by reference.

An analogue of a protease is optionally a fragment of a protease. Ananalogue of a protease is a polypeptide that has some level of activitytoward a natural or synthetic substrate of a protease. An analogueoptionally has between 0.1% and 200% the activity of a wild-typeprotease.

Specific examples of proteases illustratively have 10,000 U/g proteaseactivity or more wherein one (1) U (unit) is defined as the amount theenzyme that will liberate the non-proteinous digestion product from milkcasein (final concentration 0.5%) to give Folin's color equivalent to 1μmol of tyrosine per minute at the reaction initial reaction stage whena reaction is performed at 37° C. and pH 7.2. Illustratively, theprotease activity is anywhere between 10,000 PU/g to 1,500,000 U/ginclusive or greater. It is appreciated that lower protease activitiesare operable. Protease activity is optionally in excess of 300,000 U/g.Optionally, protease activity is between 300,000 U/g and 2,000,000 U/gor higher.

A protease is a “peptide,” “polypeptide,” and “protein” (terms usedherein synonymously) and is intended to mean a natural or syntheticcompound containing two or more amino acids having some level ofactivity toward a natural or synthetic substrate of a wild-typeprotease. A wild-type protease is a protease that has an amino acidsequence identical to that found in an organism in nature. Anillustrative example of a wild-type protease is that found at GenBankAccession No. P06874 and SEQ ID NO: 1.

A protease functions with one or more cofactor ions or proteins. Acofactor ion is illustratively a zinc, cobalt, or calcium.

Methods of screening for protease activity are known and standard in theart. Illustratively, screening for protease activity in a proteaseprotein or analogue thereof illustratively includes contacting aprotease or analogue thereof with a natural or synthetic substrate of aprotease and measuring the enzymatic cleavage of the substrate.Illustrative substrates for this purpose include casein of which iscleaved by a protease to liberate folin-positive amino acids andpeptides (calculated as tyrosine) that are readily measured bytechniques known in the art. The synthetic substrate furylacryloylatedtripeptide 3-(2-furylacryloyl)-L-glycyl-L-leucine-L-alanine obtainedfrom Sachem AG, Bubendorf, Switzerland is similarly operable.

Amino acids present in a protease or analog thereof include the commonamino acids alanine, cysteine, aspartic acid, glutamic acid,phenylalanine, glycine, histidine, isoleucine, lysine, leucine,methionine, asparagine, praline, glutamine, arginine, serine, threonine,valine, tryptophan, and tyrosine; as well as less common naturallyoccurring amino acids, modified amino acids or synthetic compounds, suchas alpha-asparagine, 2-aminobutanoic acid or 2-aminobutyric acid,4-aminobutyric acid, 2-aminocapric acid (2-aminodecanoic acid),6-aminocaproic acid, alpha-glutamine, 2-aminoheptanoic acid,6-aminohexanoic acid, alpha-aminoisobutyric acid (2-aminoalanine),3-aminoisobutyric acid, beta-alanine, allo-hydroxylysine,allo-isoleucine, 4-amino-7-methylheptanoic acid,4-amino-5-phenylpentanoic acid, 2-aminopimelic acid,gamma-amino-beta-hydroxybenzenepentanoic acid, 2-aminosuberic acid,2-carboxyazetidine, beta-alanine, beta-aspartic acid, biphenylalanine,3,6-diaminohexanoic acid, butanoic acid, cyclobutyl alanine,cyclohexylalanine, cyclohexylglycine, N5-aminocarbonylomithine,cyclopentyl alanine, cyclopropyl alanine, 3-sulfoalanine,2,4-diaminobutanoic acid, diaminopropionic acid, 2,4-diaminobutyricacid, diphenyl alanine, N,N-dimethylglycine, diaminopimelic acid,2,3-diaminopropanoic acid, S-ethylthiocysteine, N-ethylasparagine,N-ethylglycine, 4-aza-phenylalanine, 4-fluoro-phenylalanine,gamma-glutamic acid, gamma-carboxyglutamic acid, hydroxyacetic acid,pyroglutamic acid, homoarginine, homocysteic acid, homocysteine,homohistidine, 2-hydroxyisovaleric acid, homophenylalanine, homoleucine,homoproline, homoserine, homoserine, 2-hydroxypentanoic acid,5-hydroxylysine, 4-hydroxyproline, 2-carboxyoctahydroindole,3-carboxylsoquinoline, isovaline, hydroxypropanoic acid (lactic acid),mercaptoacetic acid, mercaptobutanoic acid, sarcosine,4-methyl-3-hydroxyproline, mercaptopropanoic acid, norleucine, nipecoticacid, nortyrosine, norvaline, omega-amino acid, ornithine, penicillamine(3-mercaptovaline), 2-phenylglycine, 2-carboxypiperidine, sarcosine(N-methylglycine), 2-amino-3-(4-sulfophenyl)propionic acid,1-amino-1-carboxycyclopentane, 3-thienylalanine,epsilon-N-trimethyllysine, 3-thiazolylalanine, thiazolidine 4-carboxylicacid, alpha-amino-2,4-dioxopyrimidinepropanoic acid, and2-naphthylalanine. A lipase includes peptides having between 2 and about1000 amino acids or having a molecular weight in the range of about150-350,000 Daltons.

A protease is obtained by any of various methods known in the artillustratively including isolation from a cell or organism, chemicalsynthesis, expression of a nucleic acid sequence, and partial hydrolysisof proteins. Chemical methods of peptide synthesis are known in the artand include solid phase peptide synthesis and solution phase peptidesynthesis or by the method of Hackeng, T M, et al., Proc Natl Acad SciUSA, 1997; 94(15):7845-50, the contents of which are incorporated hereinby reference. A protease may be a naturally occurring or non-naturallyoccurring protein. The term “naturally occurring” refers to a proteinendogenous to a cell, tissue or organism and includes allelicvariations. A non-naturally occurring peptide is synthetic or producedapart from its naturally associated organism or modified and is notfound in an unmodified cell, tissue or organism.

Modifications and changes can be made in the structure of a protease andstill obtain a molecule having similar characteristics as protease(e.g., a conservative amino acid substitution). For example, certainamino acids can be substituted for other amino acids in a sequencewithout appreciable loss of activity or optionally to reduce or increasethe activity of an unmodified protease. Because it is the interactivecapacity and nature of a polypeptide that defines that polypeptide'sbiological functional activity, certain amino acid sequencesubstitutions can be made in a polypeptide sequence and neverthelessobtain a polypeptide with like or other desired properties.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art. It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar biologicalactivity. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. Those indicesare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine(+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant polypeptide, whichin turn defines the interaction of the polypeptide with other molecules,such as enzymes, substrates, receptors, antibodies, antigens, and thelike. It is known in the art that an amino acid can be substituted byanother amino acid having a similar hydropathic index and still obtain afunctionally equivalent polypeptide. In such changes, the substitutionusing amino acids whose hydropathic indices are within ±2, those within±1, and those within ±0.5 are optionally used.

Substitution of like amino acids can also be made on the basis ofhydrophilicity. The following hydrophilicity values have been assignedto amino acid residues: arginine (+3.0); lysine (+3.0); aspartate(+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2);glutamine (+0.2); glycine (0); proline (−0.5±1); threonine (−0.4);alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3);valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5); tryptophan (−3.4). It is understood that an aminoacid can be substituted for another having a similar hydrophilicityvalue and still obtain an enzymatically equivalent polypeptide. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2, those within ±1, and those within ±0.5 are optionally used.

Amino acid substitutions are optionally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. Exemplarysubstitutions that take various of the foregoing characteristics intoconsideration are well known to those of skill in the art and include(original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys),(Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly:Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg),(Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe),and (Val: Ile, Leu). Embodiments of this disclosure thus contemplatefunctional or biological equivalents of a polypeptide as set forthabove. In particular, embodiments of the polypeptides can includeanalogues having about 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequenceidentity to a wild-type protease.

It is further appreciated that the above characteristics are optionallytaken into account when producing a protease with reduced or improvedenzymatic activity. Illustratively, substitutions in a substrate bindingsite, exosite, cofactor binding site, catalytic site, or other site in aprotease protein may alter the activity of the enzyme toward asubstrate. In considering such substitutions the sequences of otherknown naturally occurring or non-naturally occurring proteases may betaken into account. Illustratively, a corresponding mutation to that ofAsp213 in thermolysin is operable such as that done by Mild, Y, et al.,Journal of Molecular Catalysis B: Enzymatic, 1996; 1:191-199, thecontents of which are incorporated herein by reference. Optionally, asubstitution in thermolysin of L144 such as to serine alone or alongwith substitutions of G8C/N60C/S65P are operable to increase thecatalytic efficiency by 5-10 fold over the wild-type enzyme. Yasukawa,K, and Inouye, K, Biochimica et Biophysica Acta (BBA)—Proteins &Proteomics, 2007; 1774:1281-1288, the contents of which are incorporatedherein by reference. The mutations in the bacterial neutral proteasefrom Bacillus stearothermophilus of N116D, Q119R, D150E, and Q225R aswell as other mutations similarly increase catalytic activity. De Kreig,A, et al., J. Biol. Chem., 2002; 277:15432-15438, the contents of whichare incorporated herein by reference, De Kreig also teach severalsubstitutions including multiple substitutions that either increase ordecrease the catalytic activity of the protease. Id. and De Kreig, Eur JBiochem, 2001; 268(18):4985-4991, the contents of which are incorporatedherein by reference. Other substitutions at these or other sitesoptionally similarly affect enzymatic activity. It is within the levelof skill in the art and routine practice to undertake site directedmutagenesis and screen for subsequent protein activity such as by themethods of De Kreig, Eur J Biochem, 2001; 268(18):4985-4991 for whichthis reference is similarly incorporated herein by reference.

A protease is illustratively recombinant. Methods of cloning,synthesizing or otherwise obtaining nucleic acid sequences encoding aprotease are known and standard in the art that are equally applicableto lipase. Similarly, methods of cell transfection and proteinexpression are similarly known in the art and are applicable herein.Exemplary cDNA encoding the protein sequence of SEQ ID NO: 1 is thenucleotide sequence found at accession number M11446 and SEQ ID NO: 2.

A protease may be coexpressed with associated tags, modifications, otherproteins such as in a fusion peptide, or other modifications orcombinations recognized in the art. Illustrative tags include 6×His,FLAG, biotin, ubiquitin, SUMO, or other tag known in the art. A tag isillustratively cleavable such as by linking to lipase or an associatedprotein via an enzyme cleavage sequence that is cleavable by an enzymeknown in the art illustratively including Factor Xa, thrombin, SUMOstarprotein as obtainable from Lifesensors, Inc., Malvern, Pa., or trypsin.It is further appreciated that chemical cleavage is similarly operablewith an appropriate cleavable linker.

Protein expression is illustratively accomplished from transcription ofa protease nucleic acid sequence, illustratively that of SEQ ID NO: 2,translation of RNA transcribed from the protease nucleic acid sequenceor analogues thereof. An analog of a nucleic acid sequence is anysequence that when translated to protein will produce a proteaseanalogue. Protein expression is optionally performed in a cell basedsystem such as in E. coli, Hela cells, or Chinese hamster ovary cells.It is appreciated that cell-free expression systems are similarlyoperable.

It is recognized that numerous analogues of protease are operable andwithin the scope of the present invention including amino acidsubstitutions, alterations, modifications, or other amino acid changesthat increase, decrease, or not alter the function of the proteaseprotein sequence. Several post-translational modifications are similarlyenvisioned as within the scope of the present invention illustrativelyincluding incorporation of a non-naturally occurring amino acid,phosphorylation, glycosylation, addition of pendent groups such asbiotinylation, fluorophores, lumiphores, radioactive groups, antigens,or other molecules.

An inventive method uses an inventive composition that is one or moreproteases incorporated into a substrate itself or into a coating on thesubstrate. The protease enzyme is optionally non-covalently associatedand/or covalently attached to the substrate or coating material or isotherwise associated therewith such as by bonding to the surface or byintermixing with the substrate/coating material during manufacture suchas to produce entrapped protease. In some embodiments the protease iscovalently attached to the substrate or coating material either bydirect covalent interaction between the protease and one or morecomponents of the substrate or coating material or by association via alink moiety such as that described in U.S. Pat. App. Publ. No.2008/0119381, the contents of which are incorporated herein byreference.

There are several ways to associate protease with a substrate orcoating. One of which involves the application of covalent bonds.Specifically, free amine groups of the protease may be covalently boundto an active group of the substrate. Such active groups include alcohol,thiol, aldehyde, carboxylic acid, anhydride, epoxy, ester, or anycombination thereof. This method of incorporating protease deliversunique advantages. First, the covalent bonds tether the proteasespermanently to the substrate and thus place them as an integral part ofthe final composition with much less, if any at all, leakage of theprotease. Second, the covalent bonds provide extended enzyme lifetime.Over time, proteins typically lose activity because of the unfolding oftheir polypeptide chains. Chemical binding such as covalent bondingeffectively restricts such unfolding, and thus improves the proteinlife. The life of a protein is typically determined by comparing theamount of activity reduction of a protein that is free or beingphysically adsorbed with that of a protein covalently-immobilized over aperiod of time.

Proteases are optionally uniformly dispersed throughout the substratenetwork to create a substantially homogenous protein platform. In sodoing, proteases may be first modified with polymerizable groups. Themodified proteases may be solubilized into organic solvents in thepresence of surfactant, and thus engage the subsequent polymerizationwith monomers such as methyl methacrylate (MMA) or styrene in theorganic solution. The resulting composition optionally includes proteasemolecules homogeneously dispersed throughout the network.

Proteases are optionally attached to surfaces of a substrate. Anattachment of 1 proteases corresponding to approximately 100% surfacecoverage was achieved with polystyrene particles with diameters rangefrom 100 to 1000 nm.

Chemical methods of protease attachment to materials will naturally varydepending on the functional groups present in the lipase and in thematerial components. Many such methods exist. For example, methods ofattaching proteins (such as enzymes) to other substances are describedin O'Sullivan et al, Methods in Enzymology, 1981; 73:147-166 andErlanger, Methods in Enzymology, 1980; 70:85-104, each of which areherein incorporated herein by reference.

Proteases are optionally present in a coating that is layered upon asubstrate wherein the protease is optionally entrapped in the coatingmaterial, admixed therewith, modified and integrated into the coatingmaterial or layered upon a coating similar to the mechanisms describedfor interactions between a protease and substrate material.

Materials operable for interactions with a protease to form an activesubstrate or coating illustratively include organic polymeric materials.The combination of these materials and a protease form a protein-polymercomposite material that is used as a substrate material or a coating.

Methods of preparing protein-polymer composite materials illustrativelyinclude use of aqueous solutions of protease and non-aqueous organicsolvent-borne polymers to produce bioactive organic solvent-borneprotein-polymer composite materials.

Methods of preparing protein-polymer composite materials areillustratively characterized by dispersion of protease in solvent-borneresin prior to curing and in the composite materials, in contrast toforming large aggregates of the bioactive proteins which diminish thefunctionality of the proteases and protein-polymer composite materials.Proteases are optionally dispersed in the protein-polymer compositematerial such that the lipases are unassociated with other bioactiveproteins and/or form relatively small particles of associated proteins.Illustratively, the average particle size of lipase particles in theprotein-polymer composite material is less than 10 μm (average diameter)such as in the range of 1 nm to 10 μm, inclusive.

Curable protein-polymer compositions are optionally two-componentsolvent-borne (2K SB) compositions. Optionally, one component systems(1K) are similarly operable. Illustratively, a protease is entrapped ina coating material such as a latex or enamel paint, varnish,polyurethane gels, or other coating materials. Illustrative examples ofincorporating enzymes into paints are presented in U.S. Pat. No.5,998,200, the contents of which are incorporated herein by reference.

In two-component systems the two components are optionally mixed shortlybefore use, for instance, application of the curable protein-polymercomposition to a substrate to form a protease containing coating such asa bioactive clear coat. Generally described, the first componentcontains a crosslinkable polymer resin and the second component containsa crosslinker. Thus, the emulsion is a first component containing acrosslinkable resin and the crosslinker is a second component, mixedtogether to produce the curable protein-polymer composition.

A polymer resin included in methods and compositions of the presentinvention can be any film-forming polymer useful in coating or substratecompositions, illustratively clear coat compositions. Such polymersillustratively include, aminoplasts, melamine formaldehydes, carbamates,polyurethanes, polyacrylates, epoxies, polycarbonates, alkyds, vinyls,polyamides, polyolefins, phenolic resins, polyesters, polysiloxanes; andcombinations of any of these or other polymers.

In particular embodiments, a polymer resin is crosslinkable.Illustratively, a crosslinkable polymer has a functional groupcharacteristic of a crosslinkable polymer. Examples of such functionalgroups illustratively include acetoacetate, acid, amine, carboxyl,epoxy, hydroxyl, isocyanate, silane, vinyl, other operable functionalgroups, and combinations thereof.

Examples of organic crosslinkable polymer resins includes aminoplasts,melamine formaldehydes, carbamates, polyurethanes, polyacrylates,epoxies, polycarbonates, alkyds, vinyls, polyamides, polyolefins,phenolic resins, polyesters, polysiloxanes, or combinations thereof.

A cross linking agent is optionally included in the composition. Theparticular crosslinker selected depends on the particular polymer resinused. Non-limiting examples of crosslinkers include compounds havingfunctional groups such as isocyanate functional groups, epoxy functionalgroups, aldehyde functional groups, and acid functionality.

In particular embodiments of protein-polyurethane composite materials, apolymer resin is a hydroxyl-functional acrylic polymer and thecrosslinker is a polyisocyanate.

A polyisocyanate, optionally a diisocyanate, is a crosslinker reactedwith the hydroxyl-functional acrylic polymer according to embodiments ofthe present invention. Aliphatic polyisocyanates are preferredpolyisocyanates used in processes for making protein-polymer compositematerials for clearcoat applications such as in automotive clearcoatapplications. Non-limiting examples of aliphatic polyisocyanates include1,4-butylene diisocyanate, 1,4-cyclohexane diisocyanate,1,2-diisocyanatopropane, 1,3-diisocyanatopropane, ethylene diisocyanate,lysine diisocyanate, 1,4-methylene bis(cyclohexyl isocyanate),diphenylmethane 4,4′-diisocyanate, an isocyanurate of diphenylmethane4,4′-diisocyanate, methylenebis-4,4′-isocyanatocyclohexane,1,6-hexamethylene diisocyanate, an isocyanurate of 1,6-hexamethylenediisocyanate, isophorone diisocyanate, an isocyanurate of isophoronediisocyanate, p-phenylene diisocyanate, toluene diisocyanate, anisocyanurate of toluene diisocyanate, triphenylmethane4,4′,4″-triisocyanate, tetramethyl xylene diisocyanate, and meta-xylenediisocyanate.

Curing modalities are those typically used for conventional curablepolymer compositions.

Protease-polymer composite materials used in embodiments of the presentinvention are optionally thermoset protein-polymer composite materials.For example, a substrate or coating material is optionally cured bythermal curing. A thermal polymerization initiator is optionallyincluded in a curable composition. Thermal polymerization initiatorsillustratively include free radical initiators such as organic peroxidesand azo compounds. Examples of organic peroxide thermal initiatorsillustratively include benzoyl peroxide, dicumylperoxide, and laurylperoxide. An exemplary azo compound thermal initiator is2,2-azobisisobutyronitrile.

Conventional curing temperatures and curing times can be used inprocesses according to embodiments of the present invention. Forexample, the curing time at specific temperatures, or under particularcuring conditions, is determined by the criteria that the cross-linkerfunctional groups are reduced to less than 5% of the total presentbefore curing. Cross-linker functional groups can be quantitativelycharacterized by FT-IR or other suitable method. For example, the curingtime at specific temperatures, or under particular curing conditions,for a polyurethane protein-polymer composite of the present inventioncan be determined by the criteria that the cross-linker functional groupNCO is reduced to less than 5% of the total present before curing. TheNCO group can be quantitatively characterized by FT-IR. Additionalmethods for assessing the extent of curing for particular resins arewell-known in the art. Illustratively, curing may include evaporation ofa solvent or by exposure to actinic radiation, such as ultraviolet,electron beam, microwave, visible, infrared, or gamma radiation.

One or more additives are optionally included for modifying theproperties of the protease-polymer composite material and/or theadmixture of organic solvent and polymer resin, the aqueous lipasesolution, the emulsion, and/or the curable composition. Illustrativeexamples of such additives include a UV absorbing agent, a plasticizer,a wetting agent, a preservative, a surfactant, a lubricant, a pigment, afiller, and an additive to increase sag resistance.

A substrate or coating including a protease is illustratively anadmixture of a polymer resin, a surfactant and a non-aqueous organicsolvent, mixed to produce an emulsion. The term “surfactant” refers to asurface active agent that reduces the surface tension of a liquid inwhich it is dissolved, or that reduces interfacial tension between twoliquids or between a liquid and a solid.

Surfactants used can be of any variety including amphoteric,silicone-based, fluorosurfactants, anionic, cationic and nonionic suchas described in K. R. Lange, Surfactants: A Practical Handbook, HanserGardner Publications, 1999; and R. M. Hill, Silicone Surfactants, CRCPress, 1999, incorporated herein by reference. Examples of anionicsurfactants illustratively include alkyl sulfonates, alkylarylsulfonates, alkyl sulfates, alkyl and alkylaryl disulfonates, sulfonatedfatty acids, sulfates of hydroxyalkanols, sulfosuccinic acid esters,sulfates and sulfonates of polyethoxylated alkanols and alkylphenols.Examples of cationic surfactants include quaternary surfactants andamineoxides. Examples of nonionic surfactants include alkoxylates,alkanolamides, fatty acid esters of sorbitol or manitol, and alkylglucamides. Examples of silicone-based surfactants include siloxanepolyoxyalkylene copolymers.

When a surface which is optionally a substrate or a coated substrate, iscontacted with biological material to produce a biological stain, theprotease enzyme or combinations of enzymes contact the stain, orcomponents thereof. The contacting allows the enzymatic activity of theprotease to interact with and enzymatically alter the components of thestain improving its removal from the substrate or coating.

Enzyme containing substrates or coatings have a surface activitygenerally expressed in Units/cm². Substrates and coatings optionallyhave functional surface activities of greater than 0.0075 Units/cm². Insome embodiments surface activity is between 0.0075 Units/cm² and 0.05Units/cm² inclusive. Optionally, surface activity is between 0.0075Units/cm² and 0.1 Units/cm² inclusive. Optionally, surface activity isbetween 0.01 Units/cm² and 0.05 Units/cm² inclusive.

It is appreciated that the inventive methods of facilitating stainremoval will function at any temperature whereby the protease is active.Optionally, the inventive process is performed at 4° C. Optionally, aninventive process is performed at 25° C. Optionally, an inventiveprocess is performed at ambient temperature. It is appreciated that theinventive process is optionally performed from 4° C. to 125° C., or anysingle temperature or range therein.

The presence of protease combined with the material of a substrate or acoating on a substrate, optionally, with water or other fluidic rinsingagent, breaks down stains for facilitated removal.

Methods involving conventional biological techniques are describedherein. Such techniques are generally known in the art and are describedin detail in methodology treatises such as Molecular Cloning: ALaboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates).

Various aspects of the present invention are illustrated by thefollowing non-limiting examples. The examples are for illustrativepurposes and are not a limitation on any practice of the presentinvention. It will be understood that variations and modifications canbe made without departing from the spirit and scope of the invention.

Example 1

Production of a bacterial neutral protease from Bacillusstearothermophilus containing material operable for coating a substrate.

Materials: Freeze-dried crickets are purchased from PetSmart. Cricketbodies reportedly contain 58.3% protein. (D. Wang, et al., EntomologicSinica, 2004; 11:275-283, incorporated herein by reference.) α-Amylase,Lipase PS, Protease N, Protease A, Protin SD AY-10, B.stearothermophilus TLP (Thermoase C160), and Thermoase GL30 (lowactivity preparation of B. stearothermophilus TLP) are obtained fromAMANO Enzyme Inc. (Nagoya, JAPAN). Polyacrylate resin Desmophen A870 BA,and the hexamethylene diisocyanate (HDI) based polyfunctional aliphaticpolyisocyanate resin Desmodur N 3600 are obtained from Bayer Corp.(Pittsburgh, Pa.). The surfactant BYK-333 is obtained from BYK-Chemie(Wallingford, Conn.). 1-butanol and 1-butyl acetate are obtained fromSigma-Aldrich Co. (Missouri, USA). Aluminum paint testing panels arepurchased from Q-Lab Co. (Cleveland, USA). All other reagents involvedin the experiments are of analytical grade.

Enzyme based 2K SB PU coatings are prepared by either a draw-down methodor by spray application and used for subsequent biological stain removalexperiments. Each enzyme is dissolved in DI water to a final enzymesolution concentration of 200 mg/mL for all water borne (WB) coatings.For solvent borne (SB) enzyme prepared coatings 50 mg/mL enzyme is used.A solution of 150 ml of deionized water containing 1.5 g B.stearothermophilus TLP is first purified by ultrafiltration (molecularweight cut-off of 30 kDa, all liquids were kept on ice).

For the draw-down method or coating preparation, the surfactant BYK 333is diluted with 1-butanol to the concentration of 17% by weight. Theresin part of the 2K SB PU coating is prepared by mixing 2.1 g ofDesmophen A 870 with 0.5 mL of 1-butyl acetate and 0.1 mL surfactant ina 20 mL glass vial. The solution is mixed using a microspatula for 1 minfollowed by addition of 0.6 mL of enzyme solution (or DI water forcontrol coating without enzyme) followed by mixing for another 1 min.This solution is then poured out into a 20-mL glass vial with 0.8 g ofNA 3600 and stirred for 1 min. This formulation produces an enzymeconcentration of 6% by weight. Pre-cleaned aluminum testing panels arecoated with the enzyme containing coating material using a draw-downapplicator with a wet film thickness of 2 mils. The coating panels arebaked at 80° C. for 30 minutes and then cured at ambient temperature for7 days.

For the spray application method, coating are prepared essentially asdescribed in FIG. 1.

Example 2

Preparation of biological stains and application to coated substrate. Anexemplary schematic of an experimental procedure is provided in FIG. 2.60 g of Freeze-dried crickets are chopped into powder by a blender(Oster, 600 watt) for 10 min. The stain solution is prepared byvigorously mixing 2 g of cricket powder with 6 mL of DI water. Atemplate of uniform spacing is used to apply the stain on the coatingsurface. The cricket stains are dried at 40° C. for 5 min followed byplacing the coating panels into a glass dish and rinsing with 200 mL ofDI water under 300 rpm shaking at RT for various times. The time of thestain removal is recorded. In order to reduce random error, the time ofthe first and last drop removed are not included. The average rinsingtime of eight stain spots is averaged for stain removal time.

Example 3

Drying time affects stain removal time. Stained coated substrate panelsprepared with coatings as in Example 1 and insect stains as in Example 2are subjected to drying at 40° C. for various times. The rinsing time ofstain drops strongly depends on the drying time. The control proteasefree coating, after being dried for 5 min, produces firmly adhered staindrops that are not removed by rinsing for 3 hr (Table 1).

TABLE 1 Drying Time (min) 3 3.2 5 Average washing time (min) 2.8 4.9>180

For the B. stearothermophilus TLP containing coated panes, the rinsingtime increases with longer drying time yet at equivalent drying timesrelative to control the protease containing coating promotesdramatically improved stain removal. (Table 2).

TABLE 2 Drying Time (min) 5 10 Average washing time (min) 28.7 79.3

Example 4

Increased rinsing intensity reduces stain removal time. The panelsprepared as in Examples 1 and 2 are subjected to various rinsingintensities. Reduced rinsing time is achieved by increasing rinsingintensity for B. stearothermophilus TLP containing coatings onsubstrates (Table 3).

TABLE 3 Shaking speed (rpm) 200 250 300 Average washing time (min) 56.544.4 28.7

Example 5

Coatings containing various enzymes are prepared as in Example 1. Eachcoating is analyzed for performance by measurement of average rinsingtime using a standard protocol of applying a cricket stain to a coatedsubstrate, drying for 5 min at 40° C. and rinsing in water at anintensity of 300 RPM. The control and various protease containingcoatings are also evaluated for roughness, contact angle, and gloss. Theresults are depicted in Table 4.

TABLE 4 Average Roughness Contact Gloss Coatings Washing time (μm) Angle(60°) SB control coating >3 hr 0.053 76.2 163.0 Lipase PS based SBcoating >3 hr 0.063 88.0 147.9 α-Amylase based >3 hr 0.078 80.5 148.4 SBcoating Thermoase C160 based 28 min 0.078 86.4 148.4 SB coating

B. stearothermophilus TLP based coatings have an improved self-cleaningfunction against insect body stains compared with other coatings (noenzyme, Lipase PS, and α-amylase). In addition, the coating surfaceproperties are insignificant different between the B. stearothermophilusTLP based coating and the control, Lipase PS, or α-amylase basedcoatings. These results indicate that physical characteristics of thecoatings are not differentially affecting the coating performance.

The rinsing times of each enzyme containing coating is compared. FIG. 3demonstrates comparison of a control SB coating (enzyme free, leftpanel) with a B. stearothermophilus TLP based coating (right panel).After 30 minutes of rinsing the B. stearothermophilus TLP based coatingshows significant stain removal. The control shows no significant stainremoval out to 3 hours of rinsing.

Similar results are observed comparing a B. stearothermophilus TLP basedcoating with a lipase and α-amylase based coating. In FIGS. 4 and 5respectively, lipase and α-amylase (left panels) show significantadherence of insect stains remaining for the entire three hour rinsingperiod. In contrast the B. stearothermophilus TLP based coatings showdramatic stain removal after an initial 30 min rinsing period withessentially complete stain removal by three hours.

Example 6

Affect of surface heating on protease function. Panels coated with B.stearothermophilus TLP based coatings as in Example 1 are subjected tobaking temperatures of 100° C. for 10 days followed by determination ofchange in surface enzyme activity. Proteolytic surface activity ofprotease containing coatings is determined following the method of Folinand Ciocalteau, J. Biol. Chem., 1927; 73: 627-50, incorporated herein byreference. Briefly, 1 mL of 2% (w/v) casein in sodium phosphate (0.05 M;pH 7.5) buffer solution is used as substrate together with 200 μl ofsodium acetate, 5 mM calcium acetate (10 mM; pH 7.5). The substratesolution is pre-incubated in a water bath for 3 min to reach 37° C. Thereaction is started by adding one piece of sample plate coated with B.stearothermophilus TLP based coating (1.2×1.9 cm²) followed by shakingfor 10 min at 200 rpm at which time the reaction is stopped by adding 1ml of 110 mM tricholoroacetic acid (TCA) solution. The mixture isincubated for 30 min at 37° C. prior to centrifugation. The equivalentof tyrosine in 400 μL of the TCA-soluble fraction is determined at 660nm using 200 μL of 25% (v/v) Folin-Ciocalteau reagent and 1 mL 0.5 Msodium carbonate. One unit of activity is defined as the amount ofenzyme hydrolyzing casein to produce absorbance equivalent to 1.0 mmolof tyrosine per minute at 37° C. This result is converted to Units/cm².FIG. 6 illustrates that B. stearothermophilus TLP surface activity isreduced by approximately 50% after long term high temperature baking(FIG. 6A). Coincidentally, the time of stain cleaning is increased (FIG.6B).

Example 7

Enzyme loading is titered in coatings prepared and coated onto substratepanels as in Example 1 and with insect stains applied as in Example 2 atloading concentrations of enzyme of 0.2% (A), 2.0% (B), 4.0% (C), 6.0%(D), and 8.0% (E) of thermolysin, and the thermolysin-like-proteins fromBacillis cereus, Lactobacillis sp., Bacillis megaterium,Alicyclobacillis acidocaldarious, Bacillis caldolyticus, Bacillisthermoproteolyticus, Bacillus stearothermophilus, Bacillus subtilis,Bacillus amyloliquefaciens), and Lysteria monocytogenes. The panels arebaked for 5 min at 40° C. and washed at 300 RPM for three hours.Increased protease loading correlates with increased rinsing performance(FIG. 7A-E depicting results for B. stearothermophilus TLP).

Example 8

Comparison of various protease types on insect stain removal. Coatingsare prepared as in Example 1 using protease N (bacillolysin) as aputative cysteine protease, Protin SD AY10 (subtilisin from Bacilluslicheniformis) as a putative serine protease, protease A as an exemplarymetalloprotease, and thermolysin and thermolysin-like-proteins ofExample 7, and coated onto substrates as in Example 1 with insectstaining as in Example 2. The different enzyme containing coatings arecompared after baking for 5 min at 40° C. and rinsing at 300 RPM for 3hours. Surprisingly, only the thermolysin based coatings show thedramatically improved self-cleaning function which is not observed bycoatings including, a serine protease, a cysteine protease, or anothermetalloprotease. (Table 5 and FIG. 8.)

TABLE 5 Average Protease Washing Roughness Contact Gloss Coatings Grouptime (μm) Angle (60°) Protease N Cysterine >3 hr 0.044 76.6 158.3 basedprotease SB coating (150 kU/mg) Protin SD Serine protease >3 hr 0.04076.9 159.5 AY-10 based (90 kU/mg) SB coating Protease Metalloprotease >3hr 0.040 75.7 162.4 A based (20 kU/mg) SB coating ThermoaseMetalloprotease 12 min 0.043 76.0 163.0 C160 based (1600 kU/mg) SBcoating

Example 9

Test panels are prepared as in Example 1 and are mounted onto the frontbumpers of test vehicles. A schematic of a road-test protocol isillustrated in FIG. 9. Real-life insects are collected from the road bydriving. The vehicle is driven during summer evenings for ˜500 miles tocollect insect bodies. The average speed of driving is 65 mph.

Within three days of insect collection the panels are rinsed either innatural rain (driving condition) or in lab on a water bath at a rate ofshaking rate of 200 rpm. Photos are taken prior to and after the rinsingprocedure. Panels are visually checked and counted prior to, during, andafter rinsing to identify differences in stain removal from test andcontrol panels.

A clear increase in stain-removal effectiveness under mild rinsing isobserved on enzyme-containing coatings relative to control coatingswithout enzyme as is illustrated in FIGS. 10 and 11. The insect staincounts for enzyme containing coated panels at before, after 5 min, andafter 120 min of rinsing are 18, 13, and 10 respectively. The insectstain counts for enzyme control coated panels at before, after 5 min,and after 120 min of rinsing are 18, 17, and 15 respectively. The roadtest is repeated three times and the average percent remaining insectstains in enzyme containing and control coatings after rinsing forvarious times are plotted in FIG. 12. The enzyme containing coatingspromote active insect stain removal using environmentally obtainedinsects under normal road driving conditions.

Example 10

Enzyme containing coatings are prepared as in Example 1 using buffers ofpH 6.4 and pH 11. Coated aluminum plates are subjected to insectstaining as in Example 9. Enzyme containing coatings prepared at both pHlevels are superior to control (FIG. 13).

Various modifications of the present invention, in addition to thoseshown and described herein, will be apparent to those skilled in the artof the above description. Such modifications are also intended to fallwithin the scope of the appended claims.

It is appreciated that all reagents are obtainable by sources known inthe art unless otherwise specified or synthesized by one of ordinaryskill in the art without undue experimentation. Methods of nucleotideamplification, cell transfection, and protein expression andpurification are similarly within the level of skill in the art.

Patents and publications mentioned in the specification are indicativeof the levels of those skilled in the art to which the inventionpertains. These patents and publications are incorporated herein byreference to the same extent as if each individual application orpublication was specifically and individually incorporated herein byreference.

The foregoing description is illustrative of particular embodiments ofthe invention, but is not meant to be a limitation upon the practicethereof. The following claims, including all equivalents thereof, areintended to define the scope of the invention.

1. A method of facilitating the removal of a biological stain on asubstrate or a coating comprising: providing a substrate or a coating;associating a protease or analogue thereof with said substrate or saidcoating such that said protease or analogue thereof is capable ofenzymatically degrading a component of a biological stain.
 2. The methodof claim 1 wherein said protease is a bacterial neutralthermolysin-like-protease from Bacillus stearothermophilus.
 3. Themethod of claim 1 wherein the surface activity of the substrate orcoating is 0.0075 units/cm² or greater.
 4. The method of claim 1 whereinsaid protease or analogue thereof is covalently attached to saidsubstrate or to said coating.
 5. The method of claim 1 wherein saidprotease or analogue thereof is non-covalently adhered to or admixedinto said substrate or said coating.
 6. The method of claim 1 whereinsaid substrate or said coating comprises an organic crosslinkablepolymer resin.
 7. The method of claim 6 wherein said organiccrosslinkable polymer resin comprises a functional group ofacetoacetate, acid, amine, carboxyl, epoxy, hydroxyl, isocyanate,silane, vinyl, or combinations thereof.
 8. The method of claim 7 whereinsaid organic crosslinkable polymer resin is aminoplasts, melamineformaldehydes, carbamates, polyurethanes, polyacrylates, epoxies,polycarbonates, alkyds, vinyls, polyamides, polyolefins, phenolicresins, polyesters, polysiloxanes, or combinations thereof.
 9. Themethod of claim 7 wherein said organic crosslinkable polymer is ahydroxyl-functionalized acrylate resin.
 10. A composition forfacilitating biological stain removal comprising: a substrate or acoating; and a thermolysin, thermolysin-like protease, or analoguesthereof capable of degrading a biological stain component, saidthermolysin, thermolysin-like protease, or analogue thereof associatedwith said substrate or said coating.
 11. The composition of claim 10wherein said thermolysin-like protease is a bacterial neutralthermolysis-like-protease from Bacillus stearothermophilus.
 12. Thecomposition of claim 10 wherein said substrate or said coating comprisesan organic crosslinkable polymer resin having a functional group ofacetoacetate, acid, amine, carboxyl, epoxy, hydroxyl, isocyanate,silane, vinyl, or combinations thereof.
 13. The composition of claim 12wherein said organic crosslinkable polymer resin is aminoplasts,melamine formaldehydes, carbamates, polyurethanes, polyacrylates,epoxies, polycarbonates, alkyds, vinyls, polyamides, polyolefins,phenolic resins, polyesters, polysiloxanes, or combinations thereof. 14.The composition of claim 12 wherein said organic crosslinkable polymeris a hydroxyl-functionalized acrylate resin.
 15. The composition ofclaim 10 or 11 wherein said protease or analogue thereof is covalentlyassociated with said resin.
 16. The composition of claim 10 wherein saidsubstrate or said coating has a surface activity of 0.0075 units/cm² orgreater.